Producing method of synthetic collagen nano-fiber

ABSTRACT

With respect to synthetic collagen that has so far been difficult to be nano-fiberized, a method for producing uniform and long fibrous nano-fibers containing a synthetic collagen is described. The nano-fibers contain a polypeptide having a peptide fragment represented by Formula ( 1 ): 
       -(Pro-Y-Gly) n   (1),
 
     wherein Y represents hydroxyproline or proline, and n is an integar ranging from 5 to 9000. The producing method includes a step of preparing a spinning solution containing the polypeptide and a polymer, and a step of spinning with an electrospinning method using the spinning solution.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of Japan application serial no. 2012-067627, filed on Mar. 23, 2012. The entirety of the above-mentioned patent application is hereby incorporated by reference herein and made a part of this specification.

TECHNICAL FIELD

The invention relates to a synthetic collagen nano-fiber and a producing method thereof.

DESCRIPTION OF THE RELATED ART

Since a fiber having a nanometer-scale diameter (1 to 1000 nm) displays different material properties from those of a fiber having a micrometer or larger scale diameter, it is generally called a nano-fiber to be distinguished from other fibers. As the nano-fiber has a very large surface area per unit mass (i.e. specific surface area), its application to functional molecular carrier material, contact reaction material with fluids, material for clothes, industrial material products, daily life material products, environmental material products, electrodes and membranes of fuel cells and secondary cells, material for regenerative medicine, high efficiency particulate air (HEPA) filters, electronic paper, wearable cells, protective suits and so on have been considered. Also, research and development thereof in various fields has been conducted vigorously. In addition, influence of nanoscale material on cells has attracted attention, and a lot of researches related to medical material using various nano-fibers have been presented.

A method of producing such kind of nano-fiber is known as an electrospinning method, as described in Patent Document 1.

By the way, a collagen, which is a type of protein, has been widely used as a generic medical material. A natural type-I collagen molecule has a characteristic primary structure composed of repetition of three amino acid residues, Gly-X-Y (X and Y are various amino acids; in most cases, X is Pro and Y is Hyp). Three chains of this polypeptide gather in the same orientation to form a triple helix tertiary structure, so as to form a collagen fiber.

On the other hand, it has been presented that a synthetic collagen molecule, which was created as a collagen-like polypeptide and composed of a repetition structure of three amino acid residues, Pro-Y-Gly (Y: proline or hydroxyproline), also has a triple helix structure, and the synthetic collagen molecule has been researched as various functional materials, as described in Patent Document 2.

It is considered that by controlling the sequence of amino acid residues and utilizing the chemical modifiability of the amino acid residues, the nano-fiber composed of synthetic collagen could be made into a material having high functionality or multi-functionality which is absent in a natural collagen nano-fiber .

However, different from natural collagen molecules, such synthetic collagen has a simple repetitive structure composed of Pro-Y-Gly only. Thus, the synthetic collagen lacks interaction between the molecular chains of the polymeric peptide, and exhibits high water solubility. Accordingly, the formation of a natural collagen fiber-like long fiber has not yet been achieved. Even with the aforementioned electrospinning method, beads had been formed and fibrous substances had not been obtained; thus the formation of a natural collagen fiber-like long fiber has not been achieved.

PRIOR-ART DOCUMENTS Patent Documents

Patent Document 1: Japanese Patent Application Pub. No. 2007-303015

Patent Document 2: Pamphlet of International Publication No. WO 2008/075589

SUMMARY

In view of the foregoing, the invention provides a method of producing a nano-fiber with an electrospinning method using synthetic collagen molecules.

As a result of intensive research conducted by the inventors of the present application to solve the above problems, it has been found that a uniform and long fibrous nano-fiber can be obtained by spinning a synthetic collagen with an electrospinning method using a polymer as a spinning base material.

Namely, the invention is as follows.

[1] A method of producing a nano-fiber containing a polypeptide having a peptide fragment represented by Formula (1):

-(Pro-Y-Gly)_(n)-   (1),

wherein Y represents hydroxyproline or proline, and n is an integer ranging from 5 to 9000, the producing method including: a step of preparing a spinning solution containing the polypeptide and a polymer, and a step of spinning with an electrospinning method using the spinning solution.

[2] The method described in [1], wherein the concentration of the polypeptide in the spinning solution is from 0.1 wt % to 10 wt %.

[3] The method described in [1] or [2], wherein the concentration of the polymer in the spinning solution is from 0.1 wt % to 10 wt %.

[4] The method described in any one of [1] to [3], wherein the weight ratio of the polypeptide to the polymer in the spinning solution is from 10:1 to 1:40.

[5] The method described in any one of [1] to [4], wherein the polymer includes one, two or more selected from natural collagen, polyethylene glycols, polyvinyl alcohols, and polyglycolic acids.

[6] A nano-fiber containing a polymer and a peptide fragment represented by Formula (1):

-(Pro-Y-Gly)_(n)-   (1),

wherein Y represents hydroxyproline or proline, and n is an integer ranging from 5 to 9000.

According to the invention, a means for obtaining a uniform and long fibrous nano-fiber containing synthetic collagen is provided. Moreover, a nano-fiber containing the synthetic collagen that has so far been difficult to be fiberized is provided, and may serve as a raw material for providing a variety of uses including medical uses.

In order to make the aforementioned and other objects, features and advantages of this invention comprehensible, preferred embodiments accompanied with figures are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEM image photographs of the SC/NC nano-fibers of the examples (a and b: Example 1; c and d: Example 2).

FIGS. 2 a and 2 b are SEM image photographs of the NC/PGA nano-fiber of Example 3.

FIG. 3 shows SEM image photographs of the SC/PEG nano-fibers of the examples (a and b: Example 4; c and d: Example 5).

FIG. 4 shows SEM image photographs of the SC/PVA nano-fibers of the examples (a and b: Example 6; c and d: Example 7).

DESCRIPTION OF THE EMBODIMENTS

In the invention, the variety of amino acid residues is abbreviated into the following codes.

Ala: L-alanine residue

Arg: L-arginine residue

Asn: L-asparagine residue

Asp: L-aspartic acid residue

Cys: L-cysteine residue

Gln: L-glutamine residue

Glu: L-glutamic acid residue

Gly: glycine residue

His: L-histidine residue

Hyp: L-hydroxyproline residue

Ile: L-isoleucine residue

Leu: L-leucine residue

Lys: L-lysine residue

Met: L-methionine residue

Phe: L-phenylalanine residue

Pro: L-proline residue

Sar: sarcosine residue

Ser: L-serine residue

Thr: L-threonine residue

Trp: L-tryptophan residue

Tyr: L-tyrosine residue

Val: L-valine residue

Moreover, in this specification, an amino acid sequence of a peptide chain is expressed in accordance with the conventional expression that the N-terminal amino acid residue and the C-terminal one are at the left side and the right side, respectively.

The producing method of a nano-fiber of the invention includes a step of preparing a spinning solution containing a synthetic collagen and a polymer, and a step of spinning with an electrospinning method using the spinning solution. The invention is described in detail hereinafter.

<1>Synthetic Collagen

The synthetic collagen used in the method of the invention (hereinafter sometimes called “synthetic collagen”) is a polypeptide having a peptide fragment represented by the following Formula (1) (hereinafter called “poly PYG”).

-(Pro-Y-Gly)-   (1)

Here, Y represents hydroxyproline or proline. The hydroxyproline is, for example, 4Hyp, and is preferably trans-4-hydroxy-L-proline.

In addition, in Formula (1), a repetition number n is an integer ranging from 5 to 9000. By setting n within this range, the polypeptide can form a triple helix structure, and the formation of a nano-fiber becomes easy. In addition, from the viewpoint of the stability of a triple helix structure, n is more preferably from 5 to 1000, and further preferably from 10 to 500.

A polypeptide chain of the synthetic collagen in the invention may be in a linear form or has one or more branches. In cases it has a branch, the triple helix structure may be formed after the branching point, and there may be branches after that triple helix structure.

Furthermore, whether or not the polypeptide achieves a triple helix structure may be confirmed by measuring a circular dichroism spectrum with respect to a solution of the polypeptide. Specifically, in cases where a positive Cotton effect is shown at a wavelength of 220 to 230 nm and a negative Cotton effect is shown at a wavelength of 195 to 205 nm, it is considered that the polypeptide has achieved a triple helix structure.

In addition, the polypeptide chains of the synthetic collagen in the invention may be cross-linked to one another.

Although a weight average molecular weight of the synthetic collagen in the invention is not particularly limited, from the viewpoint of the preparation of the spinning solution, spinning efficiency and stability of the triple helix structure, the weight average molecular weight is preferably from 570 to 7,000,000, and more preferably from 2,850 to 300,000.

Here, the weight average molecular weight of the synthetic collagen is measured by, for example, a method disclosed in Japanese Patent Publication No. 2003-321500 by gel permeation chromatography using a column of Superdex 200 HR 10/30 (made by GE Healthcare Japan Corporation), a flow rate of 0.5 mL/min, an eluent of a 10 mM phosphate buffer (pH=7.4) containing 150 mM of NaCl, and Gel Filtration LMW Calibration Kit and Gel Filtration HMW Calibration Kit (made by GE Healthcare Japan Corporation) as molecular weight standards, or by gel permeation chromatography using a column of Superdex Peptide PE 7.5/300 (made by GE Healthcare Japan Corporation), a flow rate of 0.25 mL/min, an eluent of 10 mM phosphate buffer (pH=7.4) containing 150 mM of NaCl, Gel Filtration LMW Calibration Kit (made by GE Healthcare Japan Corporation) and human insulin as molecular weight standards, and glycine. Alternatively, the weight average molecular weight may be measured by HPLC gel permeation chromatography using a column of TSK-GEL6000PW XL-CP 8.0—300 mm (made by Tosoh Corporation), a mobile phase of 20 mM KH₂PO₄·H₃PO₄ (pH=3.0) : MeOH=8:2, a column temperature of 40° C., a flow rate of 0.5 mL/min, a detection at 215 mn by a UV monitor and a differential refractometer, and pullulan (produced by Showa Denko K. K.) having a molecular weight of 50,000 to 1,600,000 and dextran (by Polymer Standards Service GmbH) having a molecular weight of 11,900,000 as molecular weight standards. In addition, as a detector of this HPLC gel permeation chromatography, DAWN HELEOS and ptolab rEX of Wyatt Technology Corporation are used, and thus a gel permeation chromatograph/multi-angle laser light scattering (GPC-MALS) method can also be used for measurement. The weight average molecular weight of the synthetic collagen described in this specification is a value measured by these methods.

The polypeptide of the synthetic collagen in the invention may be composed of poly PYG only, but may also contain other amino acid residues or peptide fragment in addition to poly PYG, or an alkylene group, without impairing the stability of the triple helix structure and within a range of not impairing the effects of the invention.

Examples of the amino acid residues include at least one selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Sar, Ser, Thr, Trp, Tyr, and Val. Examples of the peptide fragment include a peptide obtained by combining a plurality of one of more of the aforementioned amino acid residues. While the alkylene group may be in a linear or branched form and is not particularly limited, specific examples thereof include an alkylene group of 1 to 18 carbon atoms, and practically, an alkylene group of 2 to 12 carbon atoms is preferred.

In the polypeptide of the synthetic collagen in the invention, the weight ratio of the poly PYG to the amino acid residues or peptide fragment other than poly PYG or to the alkylene group is from 1:99 to 100:0, and preferably in the range of 10:90 to 100:0.

The polypeptide of the synthetic collagen in the invention may form, within a range of not impeding spinning of the nano-fiber, a salt with an inorganic acid (hydrochloric acid or sulfuric acid, etc.), an organic acid (acetic acid, lactic acid, maleic acid, oxalic acid or citric acid, etc.), a metal (sodium or potassium, etc.), or an organic base (trimethylamine or triethylamine, etc.). The salt compounds of the polypeptide of synthetic collagen in the invention may be used alone or in combination of two or more.

The polypeptide having the poly PYG may be obtained by any possible method.

For example, the polypeptide may be obtained by, preferably, performing a condensation reaction using a peptide oligomer composed of amino acids constituting the poly PYG, which is obtained by a known solid- or liquid-phase synthesis method.

The condensation reaction of the peptide oligomer is generally performed in a solvent. Arbitrary solvent can be used as long as it is capable of dissolving (partly or wholly dissolving) or suspending the peptide oligomer as raw materials, and water and an organic solvent may be used in general. Specific examples thereof include water, amides (dimethylformamide, dimethylacetamide and hexamethylphosphoroamide, etc.), sulfoxides (dimethyl sulfoxide, etc.), nitrogen-containing cyclic compounds (N-methylpyrrolidone and pyridine, etc.), nitriles (acetonitrile, etc.), ethers (dioxane and tetrahydrofuran, etc.), alcohols (methyl alcohol, ethyl alcohol and propyl alcohol, etc.), and mixed solvents thereof. Among these solvents, water, dimethylformamide, and dimethyl sulfoxide are preferably used.

In addition, the condensation reaction of the peptide oligomer is preferably perfonned in the presence of a dehydrating agent (dehydration condensing agent, condensation assistant). Upon reacting in the presence of the dehydration condensing agent and the condensation assistant, a tedious treatment of repeating deprotection and amino acid binding is not required, and the condensation reaction smoothly proceeds while suppressing the occurrence of dimerization and cyclization.

The dehydration condensing agent is not particularly limited as long as it effectively performs dehydration condensation in the solvent. Examples thereof include carbodiimide condensing agents [diisopropylcarbodiimide (DIPC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC=WSCI), hydrochloric salt of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSCI.HCl), and dicyclohexylcarbodiimide (DCC), etc.], fluorophosphate condensing agenta [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate, benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate, and benzotriazol-1-yl-tris(dimethylamino)phosphonium hexafluorophosphide (BOP), etc.], and diphenylphosphorylazide (DPPA).

These dehydration condensing agents may be used alone or in combination of two or more thereof. Preferred examples of the dehydration condensing agent include carbodiimide condensing agents, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and hydrochloric salt of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

The amount of the dehydration condensing agent used is generally in the range of 0.7 to 5 moles, preferably 0.8 to 2.5 moles, and further preferably 0.9 to 2.3 moles (e.g., 1 to 2 moles), per mole of the total amount of the peptide fragments in cases where a nonaqueous solution containing no water is used. In a solvent containing water (aqueous solvent), in view of deactivation of the dehydration condensing agent due to water, the amount of the dehydration condensing agent used is generally in the range of 2 to 500 moles, preferably 5 to 250 moles, and further preferably 10 to 125 moles, per mole of the total amount of the peptide fragments.

The condensation assistant is not particularly limited as long as it accelerates the condensation reaction. Examples thereof include N-hydroxy polycarboxylic imides [such as N-hydroxydicarboxylic imides, e.g., N-hydroxysuccinimide (HONSu) and N-hydroxy-5-norbomene-2,3-dicarboxylic imide (HONB), etc.], N-hydroxytriazoles [such as N-hydroxybenzotriazoles, e.g., 1-hydroxybenzotriazole (HOBt), etc.], triazines such as 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOOBt), etc., and ethyl 2-hydroxyimino-2-cyanoacetate.

These condensation assistants may also be used alone or in combination of two or more thereof Preferred examples of the condensation assistant include N-hydroxydicarboxylic imides (such as HONSu, etc.), and N-hydroxybenzotriazoles (HOBt, etc.) or N-hydroxybenzotriazines.

The amount of the condensation assistant used is generally in the range of 0.5 to 5 moles, preferably 0.7 to 2 moles, and further preferably 0.8 to 1.5 mole, irrespective of the type of the solvent.

The dehydration condensing agent and the condensation assistant are preferably used in a suitable combination. Examples of the combination include DCC-HONSu (HOBt or HOOBt) and WSCI-HONSu (HOBt or HOOBt).

In the condensation reaction of the peptide oligomer, the pH value of the reaction solution may be controlled. Generally, the pH value of the reaction solution is controlled to be around neutral (a pH value of about 6 to 8). Generally, the pH value may be controlled by using an inorganic base (sodium hydroxide, potassium hydroxide, sodium carbonate, or sodium hydrogencarbonate, etc.), an organic base, an inorganic acid (hydrochloric acid, etc.), or an organic acid.

In addition, a base that does not participate in the condensation reaction may be added. Examples of such base include tertiary amines, e.g., trialkylamines such as trimethylamine, triethylamine and diisopropylethylamine, etc., and heterocyclic tertiary amines such as N-methylmorpholine and pyridine, etc. The amount of such base used is generally in the range of 1 to 2 times the total molar number of the peptide oligomer.

In the polypeptide obtained as the above, reagents used in the reaction remain. The reagents affect the spinning process in the method of the invention, and hence are preferably removed. The removal of the residual reagents is done with a known method, such as a dialysis method, a column method, an ultrafiltration method or the like.

In addition, considering the stability of and the ease of handling the polypeptide, it is preferred that a reaction solvent is replaced with a preservation solvent. The reaction solvent may be replaced with the target preservation solvent by using the latter as a dialysis external fluid in a dialysis method, or by using the latter as a mobile phase in a column method.

The preservation solvent is not particularly limited as long as it can suppress alterations in the physical properties of the resulting active ingredient polypeptide. Examples thereof include water, physiological saline and a buffer having a buffering function in a range of from weak acid to weak alkali. Nevertheless, it is preferred that no substance affecting the spinning process is applied in the method of the invention.

<2>Polymer

In the producing method of the nano-fiber of the invention, since a polymer is used as a spinning base material, it is possible to spin the synthetic collagen that that has so far been difficult to be fiberized with an electrospinning method. Consequently, uniform and long fibrous nano-fibers containing a synthetic collagen may be obtained.

The polymer in the invention is not particularly limited as long as it can be used as the spinning base material, and may be synthetic or natural. Examples thereof include polyethylene glycol, polyvinylidene fluoride, polyvinylidene fluoride-hexafluoropropylene copolymer, polyacrylonitrile, polyacrylonitrile-methacrylate copolymer, polymethyl methacrylate, polyvinyl chloride, polyvinylidene chloride-acrylate copolymer, polyethylene, polypropylene, polybenzimidazole, polyvinyl alcohol, cellulose, cellulose acetate butyrate, polypropylene oxide, polyethylene sulfide, SBS copolymer, nylon 6, nylon 66, nylon 11, nylon 12, nylon 610, nylon 612 and copolymer thereof, polyhydroxybutyrate, polyvinyl acetate, polyethylene oxide, natural collagen, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polyarylate, polypropylene fumarate, polypeptide, protein, coal-tar pitch, petroleum pitch, polybutylene succinate, polyethylene succinate, polystyrene, polycarbonate, polyhexamethylene carbonate, polyvinyl isocyanate, polybutyl isocyanate, polymethyl methacrylate, polyethyl methacrylate, poly-n-propyl methacrylate, poly-n-butyl methacrylate, polymethyl acrylate, polyethyl acrylate, polybutyl acrylate, polyethylene terephthalate, polybutylene terephthalate, polytrimethylene terephthalate, polyethylene naphthalate, polymer of these polyester resins, poly-p-phenylene terephthalamide, poly-p-phenylene terephthalamide-3,4′-oxyphenylene terephthalamide copolymer, poly-m-phenylene isophthalamide, cellulose diacetate, cellulose triacetate, methylcellulose, propyl cellulose, benzyl cellulose, fibroin, natural rubber, polyvinyl methyl ether, polyvinyl ethyl ether, polyvinyl n-propyl ether, polyvinyl isopropyl ether, polyvinyl n-butyl ether, polyvinyl isobutyl ether, polyvinyl t-butyl ether, polyvinylidene chloride, polyvinyl methyl ketone, polymethyl isopropenyl ketone, polycyclopentene oxide, polystyrene sulfone, chitin and derivative thereof, chitosan and derivative thereof, hyaluronic acid and derivative thereof, chondroitin and derivative thereof, deoxyribonucleic acid and derivative thereof, polyglutamic acid and derivative thereof, and glucomannan and derivative thereof A polymer mixture of two or more selected from these polymers may also be used. Among these, polyethylene glycol, polyvinyl alcohol, polyglycolic acid, natural collagen and so on, which have biocompatibility, are suitable for providing the resulting nano-fiber for medical use, and are thus preferred.

Although a weight average molecular weight of the polymer of the invention is not particularly limited, it is preferably from 50,000 to 1,000,000, and more preferably from 60,000 to 900,000. Furthermore, such weight average molecular weight may be measured by a gel permeation chromatograph method and a light scattering method.

<3>Solvent

The synthetic collagen and the polymer in the invention are used in the form of a spinning solution after being dissolved in a solvent.

As such solvent, it is not particularly limited as long as it is capable of dissolving the synthetic collagen and the polymer, evaporating at the spinning stage, and forming a fiber. Examples thereof include water, ethanol, methanol, isopropanol, acetone, sulfolane acetone, propanol, dichloromethane, formic acid, hexafluoroisopropanol, hexafluoroacetone, methyl ethyl ketone, chloroform, isopropanol, toluene, tetrahydrofuran, benzene, benzyl alcohol, 1,4-dioxane, carbon tetrachloride, cyclohexane, cyclohexanone, dichloromethane, phenol, pyridine, trichloroethane, acetic acid, N,N-dimethylformamide, dimethylsulfoxide, N,N-dimethylacetamide, 1-methyl-2-pyrrolidone, ethylene carbonate, propylene carbonate, dimethyl carbonate, acetonitrile, N-methylmorpholine-N-oxide, butylene carbonate, 1,4- butyrolactone, diethyl carbonate, diethyl ether, 1,2-dimethoxyethane, 1,3-dimethyl-2-imidazolidinone, 1,3-dioxolane, ethylmethyl carbonate, methyl formate, 3-methyloxazolidine-2-one, methyl propionate, 2-methyltetrahydrofuran and so on. These solvents may be used alone or in a combination of two or more thereof.

<4>Other Arbitrary Components

In the spinning solution in the method of the invention, in addition to the aforementioned necessary components, arbitrary components may be contained as long as they do not impede the spinning. Examples of such arbitrary components include an adhesive, an electrolyte, and so on.

When the adhesive is added, as the produced nano-fibers are bonded at a contact point, when the nano-fiber is obtained in the form of nonwoven fabric, the nano-fiber may be made as a soft nonwoven fabric having little scuffing caused by rubbing with a strong force. As the adhesive, it is not particularly limited as long as it is capable of bonding the produced nano-fibers and dissolving in the solvent of the spinning solution. Examples thereof include hot-melt adhesive, elastomeric adhesive, acrylic adhesive, epoxy adhesive, and vinyl adhesive, etc. The elastomeric adhesive may be exemplified by polychloroprene rubber, styrene-butadiene rubber, butyl rubber, acrylonitrile-butadiene rubber, ethylene-propylene rubber, chlorosulfonated polyethylene rubber, and epichlorohydrin rubber. In cases the adhesive is added, it is preferably added in an amount of 0.5 to 10 wt % relative to the total weight of the synthetic collagen and the polymer in the spinning solution.

By adding the electrolyte, the charge density of the surface of the spinning solution can be increased. As a result, the spinnability may be increased. As the electrolyte, it is not particularly limited as long as it is capable of dissolving and dissociating in the spinning solution. The electrolyte may be exemplified by sodium chloride, calcium chloride, magnesium chloride, sodium carbonate, sodium hydrogencarbonate, sodium dihydrogencarbonate, and magnesium carbonate. In cases where the electrolyte is added, it is expected that its added amount is within a range that does not cause the synthetic collagen or polymer in the spinning solution to salt out, and the added amount is preferably from 0.5 wt % to 10 wt % relative to the total weight of the synthetic collagen and the polymer in the spinning solution.

<5>Step of Preparing Spinning Solution

The method of the invention includes a step of dissolving the synthetic collagen and the polymer in the solvent to prepare the spinning solution. The preparing method of the spinning solution may include dissolving the synthetic collagen and the polymer in the solvent respectively and mixing up the solutions, or may include adding, in a solutions obtained by dissolving one of the synthetic collagen and the polymer in the solvent, the other of the two. In addition, in the preparation, the synthetic collagen may be properly heated and stirred as long as the synthetic collagen is not denaturated.

The concentration of the synthetic collagen in the spinning solution is preferably from 0.1 wt % to 2.0 wt %, and more preferably from 0.5 wt % to 1.0 wt %. In addition, the concentration of the polymer in the spinning solution is preferably from 0.1 wt % to 20 wt %, and more preferably from 0.5 wt % to 10 wt %. As the concentrations of the synthetic collagen and the polymer are applied within such ranges, interactions occur between the polymer chains and between the polymer and the synthetic collagen in the spinning solution, and a continuous fiber is easily formed.

The combination ratio of the synthetic collagen and the polymer in the spinning solution is preferably from 20:1 to 1:100, and more preferably from 10:1 to 1:40, by weight, in order to obtain uniform nano-fibers of a certain length.

<6>Step of Spinning Nano-fiber

The method of the invention includes spinning the nano-fiber by performing an electrospinning method using the spinning solution as described above. A fiber having a nanoscaled fine and uniform diameter may be produced by this process.

The electrospinning method may be performed by a known means. Specifically, in a state where a voltage is applied between a nozzle filled with the spinning solution and a collector (substrate), the spinning solution is discharged from the nozzle, and the fiber is collected on the collector. Conditions for the electrospinning are not particularly limited as long as they are properly adjusted according to the type of the spinning solution and the use of resulting nano-fiber. General conditions for the method of the invention include, for example, an applied voltage of 8 to 30 kV, a discharge rate of 0.01 to 1.00 mL/hr, a vertical distance of 100 to 200 mm between the nozzle and the collector, and use of a nozzle of 22 to 25 gauge. Although the spinning environment is preferably at a temperature of 10 to 25° C. and a relative humidity of 10 to 40%, a specially strict control is not required.

According to the producing method of the invention, a fiber having a diameter of 5 nm to 50 μm may be obtained. In addition, depending on the settings and adjustments of the spinning conditions, a long and continuous nano-fiber having an average length of 200 to 300 nm may be obtained. In addition, in the nano-fiber, lump-shaped beads are not contained, or are contained only in a small amount, and uniform nano-fibers are obtained.

The nano-fiber containing synthetic collagen obtained by the invention may serve as a functional material for providing a variety of applications including materials for medical uses. For example, it can be applied to synthetic collagen-containing adsorbents that utilize high affinity to biomolecules such as blood coagulation factors, and to hemostatic agents utilizing blood coagulability.

EXAMPLES

The invention will be described in more details with the examples given below. However, the invention is not limited to these examples.

The nano-fibers of Examples 1 to 7 were prepared by the electrospinning method. Those nano-fibers were observed using a scanning electron microscope (SEM). The machine used was JSM-5600 (manufactured by JEOL Ltd.), and the observation was conducted with an accelerating voltage of 20 kV.

Example 1 SC/NC (10:1) Blend Nano-Fiber

The spinning solution was prepared by dissolving a synthetic collagen (SC, produced by JNC Corporation) and a natural collagen (NC, NMP Collagen PS/IP, produced by Nippon Meat Packers, Inc.) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, produced by Wako Pure Chemical Industries, Ltd.), wherein the SC/NC/HFIP was dissolved in a concentration of 50 mg/5 mg/1 mL. A voltage of 10 kV from a high-voltage generator was applied between a 25G stainless needle and a collector (vertical distance: 150 mm). The spinning solution was filled in a syringe connected to the above needle, and was then extruded on the collector at a discharge rate of 0.1 mL/hr. Furthermore, the humidity and temperature of the spinning environment were not controlled, and the spinning was performed in a laboratory environment.

After an SEM observation was perfonned, it was known that relatively uniform nano-fibers having an average diameter of 145 nm and containing no beads or particles was obtained (FIGS. 1 a and 1 b).

Example 2 SC/NC (1:1) Blend Nano-Fiber

The spinning was performed by the same operations as in Example 1, except that the concentration of the spinning solution was adjusted to SC/NC/HFIP=50 mg/50 mg/1 mL.

According to an SEM observation, the fiber obtained was ribbon-like (FIGS. 1 c and 1 d).

Example 3 SC/PGA Blend Nano-Fiber

A spinning solution was prepared by dissolving a synthetic collagen (SC, produced by JNC Corporation) and a polyglycolic acid (PGA, produced by Sigma-Aldrich Corporation) in 1,1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, produced by Wako Pure Chemical Industries, Ltd.), wherein the SC/PGA/HFIP was dissolved in a concentration of 50 mg/10 mg/1 mL. A voltage of 12 kV from a high-voltage generator was applied between a 25G stainless needle and a collector (vertical distance: 150 mm). The spinning solution was filled in a syringe connected to the aforementioned needle, and then was extruded to the collector at a discharge rate of 0.1 mL/hr. Furthermore, the humidity and temperature of the spinning environment were not controlled, and the spinning was performed in a laboratory environment.

After an SEM observation was performed, it was known that nano-fibers having a mean diameter of 71 nm but containing a large amount of beads was obtained (FIG. 2).

Example 4 SC/PEG 500 k Blend Nano-Fiber

A spinning solution was prepared by respectively preparing an aqueous solution containing 0.5 wt % of a synthetic collagen (SC, produced by JNC Corporation) and an aqueous solution containing 10 wt % of a polyethylene glycol (PEG, weight-average molecular weight: 500,000, produced by Wako Pure Chemical Industries, Ltd.) and mixing the same with a volume ratio of SC:PEG=2:1. A voltage of 8 kV from a high-voltage generator was applied between a 25G stainless needle and an aluminium foil collector (vertical distance: 200 mm). The spinning solution was filled in a syringe connected to the above needle, then was extruded onto the collector at a discharge rate of 0.01 mL/hr, and was collected as a uniformly distributed nonwoven fabric. Furthermore, the humidity of the spinning environment was controlled by introducing dry nitrogen, and the relative humidity in the spinning was always maintained at 15% or below. In addition, the temperature of the spinning environment was not controlled, and the spinning was performed at room temperature.

After an SEM observation was performed, it was known that relatively uniform nano-fibers having an average diameter of 115 nm was obtained (FIGS. 3 a and 3 b).

Example 5 SC/PEG 900 k Blend Nano-Fiber

The spinning was performed by the same operations as in Example 4, except that the spinning solution was prepared by respectively preparing an aqueous solution containing 0.5 wt % of a synthetic collagen (SC, produced by JNC Corporation) and an aqueous solution containing 5 wt % of a polyethylene glycol (PEG, weight-average molecular weight: 900,000, produced by Sigma-Aldrich Corporation) and mixing the same with a volume ratio of SC:PEG=2:1 (volume ratio), and the discharge rate was 0.03 mL/hr.

According to an SEM observation, nano-fibers obtained have an average diameter of 87 nm and relatively uniform fiber portions but contain a large amount of spindle-shaped beads (FIGS. 3 c and 3 d).

Example 6 SC/PVA 2000 Blend Nano-Fiber

A spinning solution was prepared by respectively preparing an aqueous solution containing 0.5 wt % of a synthetic collagen (SC, produced by JNC Corporation) and an aqueous solution containing 0.5 wt % of a polyvinyl alcohol (PVA, average degree of polymerization: 2,000, degree of saponification: 98.0 mol %, produced by Wako Pure Chemical Industries, Ltd.) and mixing the same with a volume ratio of SC:PVA=1:1. A voltage of 15 kV from a high-voltage generator was applied between a 25G stainless needle and an aluminium foil collector (vertical distance: 200 mm). The spinning solution was filled in a syringe connected to the above needle, then was extruded onto the collector at a discharge rate of 0.2 mL/hr, and was collected as a uniformly distributed nonwoven fabric. Furthermore, the humidity of the spinning environment was controlled by introducing dry nitrogen, and the relative humidity in the spinning was always maintained at 15% or below. In addition, the temperature of the spinning environment was not controlled, and the spinning was performed at room temperature.

After an SEM observation was performed, it was known that relatively uniform nano-fibers having an average diameter of 213 nm and containing no beads or particles was obtained (FIGS. 4 a and 4 b).

Example 7 SC/PVA 1500 Blend Nano-Fiber

The spinning solution was prepared by respectively preparing an aqueous solution containing 0.5 wt % of a synthetic collagen (SC, produced by JNC Corporation) and an aqueous solution containing 20 wt % of a polyvinyl alcohol (PVA, average degree of polymerization: 1,500, degree of saponification: 78 to 82 mol %, produced by Wako Pure Chemical Industries, Ltd.) and mixing the same with a volume ratio of SC:PVA=1:1. A voltage of 12 kV from a high-voltage generator was applied between a 25G stainless needle and an aluminium foil collector (vertical distance: 200 mm). The spinning solution was filled in a syringe connected to the above needle, then was extruded onto the collector at a discharge rate of 0.1 mL/hr, and was collected as a uniformly distributed nonwoven fabric. Furthermore, the humidity of the spinning environment was controlled by introducing dry nitrogen, and the relative humidity in the spinning was always maintained at 15% or below. In addition, the temperature of the spinning environment was not controlled, and the spinning was performed at room temperature.

According to an SEM observation, the nano-fibers obtained are relatively uniform, have an average diameter of 201 nm and contain no beads or particles (FIGS. 4 c and 4 d).

INDUSTRIAL UTILITY

According to the invention, a means of obtaining a uniform and long fibrous nano-fiber containing a synthetic collagen is provided. In addition, a nano-fiber containing the synthetic collagen that has so far been difficult to be fiberized is provided, which may serve as a material for providing a variety of applications including medical uses. Hence, the invention is industrially very useful.

This invention has been disclosed above in the preferred embodiments, but is not limited to those. It is known to persons skilled in the art that some modifications and innovations may be made without departing from the spirit and scope of this invention. Hence, the scope of this invention should be defined by the following claims. 

What is claimed is:
 1. A method of producing a nano-fiber that contains a polypeptide having a peptide fragment represented by Formula (1): -(Pro-Y-Gly)_(n)-   (1), wherein Y represents a hydroxyproline or proline, and n is an integer ranging from 5 to 9000, the producing method comprising: preparing a spinning solution containing the polypeptide and a polymer, and spinning with an electrospinning method using the spinning solution.
 2. The method according to claim 1, wherein a concentration of the polypeptide in the spinning solution is from 0.1 wt % to 10 wt %.
 3. The method according to claim 1, wherein a concentration of the polymer in the spinning solution is from 0.1 wt % to 10 wt %.
 4. The method according to claim 1, wherein a weight ratio of the polypeptide to the polymer in the spinning solution is from 10:1 to 1:40.
 5. The method according to claim 1, wherein the polymer comprises one, two or more selected from the group consisting of natural collagen, polyethylene glycols, polyvinyl alcohols, and polyglycolic acids.
 6. A nano-fiber, containing a polymer and a peptide fragment represented by Formula (1): -(Pro-Y-Gly)_(n)-   (1), wherein Y represents hydroxyproline or proline, and n is an integer ranging from 5 to
 9000. 